The development of new therapeutic drugs against the virus causing Severe Acute Respiratory Syndrome (SARS) is a clear imperative. Since its report in Guangdong Province, China, in November, 2002, SARS spread to other Asian countries, North America and Europe. By July 2003 there were more than 8,000 cases in 26 countries on five continents, with 774 deaths and enormous economic damage. A recently discovered coronavirus has been identified as the etiological agent for SARS. Thus far, no effective therapy exists for this virus. The replicase polyprotein is proteolytically processed by two viral proteases, papain-like protease 2 (PLpro) and 3CLpro. Proteolytic processing is essential for generating a functional replication complex, and thus the SARS-CoV proteases are attractive targets for the development of antiviral drugs that will inhibit viral replication. Building on a well-established collaborative network among the investigators participating in this project, we propose an integrated approach toward the development of new antiviral protease inhibitors. We will use a combination of strategies, beginning with characterization of proteases and viral proteins essential to assembly of the replication process, protease enzymatic characterization and X-ray determination of protease 3D molecular structures, utilization of diverse chemical libraries for initial lead discovery, structure-based drug design, synthesis of lead compounds and their optimization, followed by cell culture testing. An important strength of the application is the broad range of the participants' expertise, including virology, enzymology, structural biology of macromolecules, computer-assisted drug design, synthetic medicinal chemistry, and cell culture testing. Project 1 will characterize PLpro and evaluate the roles of PLpro and 3CLpro in proteolytic processing. Project 2 has already developed an activity assay for 3CLpro and crystallized 3CLpro with inhibitors, and will continue to develop enzymatic profiles and X-ray crystal structures of 3CLpro and PLP2 and their interactions with inhibitors. Project 3 will utilize structure-based design and high throughput screening to develop inhibitors of 3CLpro and PLP2; initial inhibitors exhibit significant activity against 3CLpro. Two scientific cores will support these projects: A protein expression core will provide proteins for target evaluation and structure-based design. A computational and high throughput screening core will provide bioinformatic support for mutational analysis, enzymatic characterization, computational support for structure based design and screening for lead inhibitors. An administrative core will provide fiscal management and administrative support.